The invention relates to the fields of photometry, spectrophotometry, fluorometry, spectrofluorometry and the like and their use in optically quantitating and or characterizing liquids and solutions.
This invention relates to the field of spectrophotometers and related instruments. More particularly the invention relates to ultra low volume instruments working in the volume range of 2 microliters or less. Such devices are particularly useful in quantitation of biotechnology samples including nucleic acids or proteins where it is desirable to keep sample loss and/or cross-contamination to a minimum.
Robertson, in International Publication Number WO 01/14855, discloses method and apparatus for liquid photometry on extremely small samples. Robertson is the inventor of the instant invention which is an improvement thereon The disclosure, of the publication is incorporated herein by reference. A U.S. nation stage application was entered into for that reference and has issued as U.S. Pat. No. 6,628,382. The prior art to WO 01/14855 A1 contains examples of attempts to supply low volume instruments. World Precision Instruments of Sarasota, Fla. offers parts from which an instrument handling less than 20 microliters can be built for around $3000. This uses a fiber optic dipping probe with a tip diameter of 1.5 mm (Dip Tip(copyright)), their miniature fiber optic spectrometer and F-O-Lite H light source. With a deuterium lights source (D2Lux) a UV spectrophotometer can be constructed.
U.S. Pat. No. 4,643,580 to Gross et al. discloses a photometer head in which there is a housing for receiving and supporting small test volumes. A fiber optic transmitter and receiver are spaced within the housing so that a drop can be suspended between the two ends.
McMillan, in U.S. Pat. No. 4,910,402, discloses apparatus in which a syringe drops liquid into the gap between two fixed fibers and an IR pulse from a LED laser is fed through the droplet. The output signal is analyzed as a function of the interaction of the radiation with the liquid of the drop.
Ocean Optics, of Dunedin, Fla. 34698 supplies a SpectroPipetter for microliter-volume samples using a sample volume of about 2 microliters. The optics not only carry light to and from the samples but act as a plunger to load the sample. The tip of the pipette includes the sample cell.
Liquids, mixtures, solutions and reacting mixtures are often characterized using optical techniques such as photometry, spectrophotometry, fluorometry, or spectrofluorometry. In order to characterize samples of these liquids, the liquid is usually contained in a vessel referred to as a cell or cuvette two or more of whose sides are of optical quality and permit the passage of those wavelengths needed to characterize the liquid contained therein. When dealing with very small sample volumes of say from 1 to 2 microliters, it is difficult to create cells or cuvettes small enough to to be filled and permit the industry standard 1 cm optical path to be used. It is also difficult and/or time consuming to clean these cells or cuvettes for use with another sample. In the case of photometry or spectrophotometry, the value most commonly sought is the sample absorbance A defined by
A=xe2x88x92log T
Where T is the transmittance, or
A=log(l/l0)
where l0 is the level of light transmitted through a blank sample (one containing all components except the one being measured or one whose absorbance is known to be negligible and with optical properties identical to those of the sample being measured), and l the level of light transmitted through the sample being measured. Most commonly the absorbance value is measured in a cell or cuvette with a 1 cm path length. However, Lambert""s Law states that for a collimated (all rays approximately parallel) beam of light passing through a homogeneous solution of uniform concentration the absorbance is proportional to the path length through the solution. For two path lengths X and Y,
(Absorbance x)/(Absorbance y)=(Pathlength x)/(Pathlength y)
Thus it is reasonable that absorbance can be measured with path lengths other than 1 cm and corrected for path length to the equivalent value for a 1 cm path which can be more easily compared to data from other spectrophotometers. The sample path lengths in the range of 0.2 to 2 mm used in this Invention can be used to generate absorbance values that can be easily corrected to the 1 cm path equivalent.
Although liquids confined by surface tension and an optical surface are well known, e.g. raindrops on the window, establishing a collimated optical light path of known length through such confined liquids has been perceived as difficult. The recent advent of small spectrometers designed to be used with fiber optics has made it possible to consider spectrophotometric geometries not readily possible before.
This invention uses the surface tension of a microliter or submicroliter sample of liquid to provide sufficient means to confine it within the analysis region of an optical analysis instrument and to carry out the requisite measurement.
The invention is an optical instrument for photometric, spectrophotometric, fluorometric or spectrofluorometric analysis of liquids contained between two substantially parallel surfaces on anvils spaced apart a known distance, wherein the sample liquid is confined by the surfaces and the surface tension of the liquid. At least two optical fibers penetrate these surfaces. One fiber is the source and the other the receiver. Ordinarily each of the surfaces contains an optical fiber. These fibers are mounted coaxially with and perpendicular to the parallel confining surfaces. The shape and nature of the surfaces serve to confine the liquid so as to center the confined droplet in the optical path of the optical fibers imbedded in the surfaces. An apparatus supporting the surfaces permits the surfaces to be controllably separated tho allow loading of the sample and cleaning of the surfaces after sample analysis as well as to pull a column of controlled length in the sample to set the optical path length during measurement. The surfaces can be separated enough that both surfaces can have a nanodrop applied so that mixing or reacting can be carried out and observed. The surfaces can be moved into a close position for sample compression as a wetting aid or, in some instances, as an alternate loading position.
For some applications, the optical fibers can be replaced by miniature sources like light emitting diodes (LEDs) and detectors or detectors with optical filters. The LEDs with their characteristically small emitting area would replace the source fiber and small solid state detectors with associated filters like those used in color charge coupled devices (CCDs) for imaging would replace the receiving fiber and spectrometer.